Project Summary Responses of vascular endothelial cells (ECs) to hemodynamic forces play significant roles in the regulation of vascular homeostasis. In vivo studies have shown that the ECs in branch points of the arterial tree are exposing to disturbed flow (DF) and express pro-inflammatory and pro-atherogenic phenotypes. In contrast, ECs in the straight part of the arterial tree are exposed to laminar shear flow (LF) and are generally spared from atherosclerosis. We hypothesize that atheroprone and atheroprotective flows activate ECs with differential spatiotemporal characteristics at subcellular levels to trigger different cellular responses. We propose to use genetically encoded biosensors based on fluorescent proteins (FPs) and fluorescence resonance energy transfer (FRET) to visualize molecular activities in individual live cells with unprecedented spatiotemporal resolution. We will study the signals relays across the plasma membrane, between neighboring cells, as well as intracellular cytosol-nuclei transitions to understand the temporal and spatial dynamics of mechanotransduction. In order to achieve effectiveness of the biosensor studies, we will incorporate a new mOrange2-mCherry FRET pair together with the CFP-YFP pair to simultaneously monitor two different molecular events in the same live cell. We will further integrate fluorescence lifetime imaging microscopy (FLIM) to simultaneously visualize multiple molecular signals across the plasma membrane, between cells, and inside the cell body, with the use of correlative FRET imaging microscopy (CFIM) developed in our labs. Three specific aims are proposed: 1) To visualize the spatiotemporal mechanotransduction across the plasma membrane: the extracellular shear stress (shear sensors) and intracellular molecular signals (transmembrane TRPC6 and Src activities at different membrane microdomains) will be simultaneously monitored under different flows to elucidate the roles of microdomains and molecular elements at the plasma membrane. 2) To dissect the role of TRPC6 in the regulation of adherent junctions (AJs) under different flows: an -catenin biosensor will be used to monitor the mechanical tension at AJs and its interplays with extra-/inter-cellular calcium ion concentrations. 3) To decipher the membrane-cytosol-nucleus ERK signaling for MCP-1 gene regulation: differential flow-regulations of the cytosolic and nucleic ERK FRET biosensors will be determined to reconstruct the spatiotemporal activation map of ERK in relation to MCP-1 gene expression. The results obtained from these studies will allow us to generate spatiotemporal correlation maps of molecular transductions/interactions and assess the roles of membrane microdomains/elements in regulating these events. These findings will provide novel understanding of the spatiotemporal basis of the molecular and mechanical mechanisms of atherosclerosis, a major pathophysiological event in cardiovascular diseases.